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Poeschl, Y.; Möller, B.; Müller, L.; Bürstenbinder, K. User-friendly assessment of pavement cell shape features with PaCeQuant: Novel functions and tools Methods Cell Biol (2020) DOI: 10.1016/bs.mcb.2020.04.010

Leaf epidermis pavement cells develop complex jigsaw puzzle-like shapes in many plant species, including the model plant Arabidopsis thaliana. Due to their complex morphology, pavement cells have become a popular model system to study shape formation and coordination of growth in the context of mechanically coupled cells at the tissue level. To facilitate robust assessment and analysis of pavement cell shape characteristics in a high-throughput fashion, we have developed PaCeQuant and a collection of supplemental tools. The ImageJ-based MiToBo plugin PaCeQuant supports fully automatic segmentation of cell contours from microscopy images and the extraction of 28 shape features for each detected cell. These features now also include the Largest Empty Circle criterion as a proxy for mechanical stress. In addition, PaCeQuant provides a set of eight features for individual lobes, including the categorization as type I and type II lobes at two- and three-cell junctions, respectively. The segmentation and feature extraction results of PaCeQuant depend on the quality of input images. To allow for corrections in case of local segmentation errors, the LabelImageEditor is provided for user-friendly manual postprocessing of segmentation results. For statistical analysis and visualization, PaCeQuant is supplemented with the R package PaCeQuantAna, which provides statistical analysis functions and supports the generation of publication-ready plots in ready-to-use R workflows. In addition, we recently released the FeatureColorMapper tool which overlays feature values over cell regions for user-friendly visual exploration of selected features in a set of analyzed cells.
Bücher und Buchkapitel

Möller, B.; Bürstenbinder, K. Semi-Automatic Cell Segmentation from Noisy Image Data for Quantification of Microtubule Organization on Single Cell Level 199-203, (2019) ISBN: 978-1-5386-3640-4 DOI: 10.1109/ISBI.2019.8759145

The structure of the microtubule cytoskeleton provides valuable information related to morphogenesis of cells. The cytoskeleton organizes into diverse patterns that vary in cells of different types and tissues, but also within a single tissue. To assess differences in cytoskeleton organization methods are needed that quantify cytoskeleton patterns within a complete cell and which are suitable for large data sets. A major bottleneck in most approaches, however, is a lack of techniques for automatic extraction of cell contours. Here, we present a semi-automatic pipeline for cell segmentation and quantification of microtubule organization. Automatic methods are applied to extract major parts of the contours and a handy image editor is provided to manually add missing information efficiently. Experimental results prove that our approach yields high-quality contour data with minimal user intervention and serves a suitable basis for subsequent quantitative studies.
Bücher und Buchkapitel

Möller, B.; Poeschl, Y.; Klemm, S.; Bürstenbinder, K. Morphological Analysis of Leaf Epidermis Pavement Cells with PaCeQuant (Cvrčková, F. & Žárský, V., eds.). Methods Mol Biol 1992, 329-349, (2019) ISBN: 978-1-4939-9469-4 DOI: 10.1007/978-1-4939-9469-4_22

Morphological analysis of cell shapes requires segmentation of cell contours from input images and subsequent extraction of meaningful shape descriptors that provide the basis for qualitative and quantitative assessment of shape characteristics. Here, we describe the publicly available ImageJ plugin PaCeQuant and its associated R package PaCeQuantAna, which provides a pipeline for fully automatic segmentation, feature extraction, statistical analysis, and graphical visualization of cell shape properties. PaCeQuant is specifically well suited for analysis of jigsaw puzzle-like leaf epidermis pavement cells from 2D input images and supports the quantification of global, contour-based, skeleton-based, and pavement cell-specific shape descriptors.
Bücher und Buchkapitel

Möller, B.; Zergiebel, L.; Bürstenbinder, K. Quantitative and Comparative Analysis of Global Patterns of (Microtubule) Cytoskeleton Organization with CytoskeletonAnalyzer2D (Cvrčková, F. & Žárský, V., eds.). Methods Mol Biol 1992, 151-171, (2019) ISBN: 978-1-4939-9469-4 DOI: 10.1007/978-1-4939-9469-4_10

The microtubule cytoskeleton plays important roles in cell morphogenesis. To investigate the mechanisms of cytoskeletal organization, for example, during growth or development, in genetic studies, or in response to environmental stimuli, image analysis tools for quantitative assessment are needed. Here, we present a method for texture measure-based quantification and comparative analysis of global microtubule cytoskeleton patterns and subsequent visualization of output data. In contrast to other approaches that focus on the extraction of individual cytoskeletal fibers and analysis of their orientation relative to the growth axis, CytoskeletonAnalyzer2D quantifies cytoskeletal organization based on the analysis of local binary patterns. CytoskeletonAnalyzer2D thus is particularly well suited to study cytoskeletal organization in cells where individual fibers are difficult to extract or which lack a clearly defined growth axis, such as leaf epidermal pavement cells. The tool is available as ImageJ plugin and can be combined with publicly available software and tools, such as R and Cytoscape, to visualize similarity networks of cytoskeletal patterns.
Publikation

Mitra, D.; Klemm, S.; Kumari, P.; Quegwer, J.; Möller, B.; Poeschl, Y.; Pflug, P.; Stamm, G.; Abel, S.; Bürstenbinder, K. Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana J Exp Bot 70, 529-543, (2019) DOI: 10.1093/jxb/ery395

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles of calcium in regulation of PC morphogenesis. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs.
Publikation

Iglesias, M. J.; Terrile, M. C.; Correa-Aragunde, N.; Colman, S. L.; Izquierdo-Álvarez, A.; Fiol, D. F.; París, R.; Sánchez-López, N.; Marina, A.; Calderón Villalobos, L. I. A.; Estelle, M.; Lamattina, L.; Martínez-Ruiz, A.; Casalongué, C. A. Regulation of SCFTIR1/AFBs E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling Redox Biol 18, 200-210, (2018) DOI: 10.1016/j.redox.2018.07.003

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1–cullin–F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction.
Publikation

Bagchi, R.; Melnyk, C. W.; Christ, G.; Winkler, M.; Kirchsteiner, K.; Salehin, M.; Mergner, J.; Niemeyer, M.; Schwechheimer, C.; Calderón Villalobos, L. I. A.; Estelle, M. The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases. EMBO J 37, 255-268, (2018) DOI: 10.15252/embj.201797159

The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.
Publikation

García, M. L.; Bó, E. D.; da Graça, J. V.; Gago-Zachert, S.; Hammond, J.; Moreno, P.; Natsuaki, T.; Pallás, V.; Navarro, J. A.; Reyes, C. A.; Luna, G. R.; Sasaya, T.; Tzanetakis, I. E.; Vaira, A. M.; Verbeek, M.; ICTV Report Consortium Corrigendum: ICTV Virus Taxonomy Profile: Ophioviridae J Gen Virol 99, 949-949, (2018) DOI: 10.1099/jgv.0.001093

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Preprints

Mitra, D.; Kumari, P.; Quegwer, J.; Klemm, S.; Moeller, B.; Poeschl, Y.; Pflug, P.; Stamm, G.; Abel, S.; Bürstenbinder, K. Microtubule-associated protein IQ67 DOMAIN5 regulates interdigitation of leaf pavement cells in Arabidopsis thaliana bioRxiv (2018) DOI: 10.1101/268466

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis, as indicated by reduced interdigitation of neighboring cells in the leaf epidermis of iqd5 mutants. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced asymmetric expansion. Lastly, we provide evidence for IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate multi-polar growth in PCs.
Publikation

Ziegler, J.; Schmidt, S.; Strehmel, N.; Scheel, D.; Abel, S. Arabidopsis Transporter ABCG37/PDR9 contributes primarily highly oxygenated Coumarins to Root Exudation Sci Rep 7, 3704, (2017) DOI: 10.1038/s41598-017-03250-6

The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins.
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